The term "Staphylococcus aureus" as used herein, refers to bacteria classified as such in Bergey's Manual of Systematic Bacteriology (P. H. A. Sneath, ed., 1986, PP. 1015-1019, Williams & Wilkins). Detection of Staphylococcus aureus is important in various medical and public health contexts. In particular, Staphylococcus aureus can cause severe food poisoning. Thousands of cases of food poisoning are reported in the United States each year. Many more unreported cases are suspected. Foods are examined for the presence of S. aureus and/or its enterotoxins to confirm that S. aureus is the causative agent of foodborne illness, to determine whether a food is a potential source of "staph" food poisoning, and to demonstrate post-processing contamination, which generally is due to human contact or contaminated food-contact surfaces.
Methods for detecting and enumerating Staphylococcus aureus depend to some extent on the reasons for testing the food and on the past history of the test material itself. The methods of analysis for S. aureus which are most commonly used (and which provide the type of information required by the Food and Drug Administration) are given in Chapters 14 and 15 of the FDA/BAM Bacteriological Analytical Manual (6th edition, 1984, Association of Official Analytical Chemists). Generally, such methods involve the isolation of Staphylococcus aureus from an appropriately prepared sample on microbiological medium under conditions favorable for growth of these bacteria. The resulting colonies then typically are examined for morphological and biochemical characteristics, a process that generally is initiated 48 hours after acquisition of the sample and disadvantageously takes between four to six days to complete. Therefore, it is an aspect of the present invention to provide nucleic acid probes which are specific for Staphylococcus aureus and which do not react with other bacteria or fungi which may be present in sampled materials. Such probes may be used in a variety of assay systems which avoid many of the disadvantages associated with traditional, multi-day culturing techniques.
It is another aspect of the present invention to provide probes which can hybridize to target regions which can be rendered accessible to probes under normal assay conditions.
While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make Staphylococcus aureus specific probes.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantifying such homology levels. Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to Staphylococcus aureus and in particular, do not provide Staphylococcus aureus specific probes useful in assays for detecting Staphylococcus aureus in food and other samples.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, at least in E. coli, are referred to as 5S, 16S and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see below--Hybridization) to target nucleic acid sequences. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to carry detection ligands (e.g. fluorescien, 32-P, biotin, etc.), or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails"). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds.
The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.